Dual conjugation process for preparing antibody-drug conjugates

ABSTRACT

The present invention relates to a process for preparing antibody-drug conjugates and to antibody-drug conjugates wherein therapeutic moieties are conjugated to one or more engineered cysteines as well as to one or more reduced interchain cysteines via a cleavable or non-cleavable linker.

FIELD OF THE INVENTION

The present invention relates to a new process for conjugatingtherapeutic moieties via a cleavable or non-cleavable linker toantibodies. Furthermore, it relates to new antibody-drug conjugates.

BACKGROUND OF THE PRESENT INVENTION

Antibody-drug conjugates (ADCs) are an emerging class of targetedtherapeutics, in which the specificity of antibodies and the potency ofcytotoxic molecules are combined.

Cysteine-engineered antibodies are increasingly used for conjugation ofa therapeutic moiety (e.g. a drug, toxin, or a chelator for aradioactive isotope), a fluorescent label or a hydrophilic polymer. Theintroduction of a cysteine residue at a suitable position of theantibody allows control of the site of conjugation and the obtainedsite-specific conjugates are more homogeneous than the conjugatesobtained via wild-type conjugation, i.e. conjugation via reducedinterchain cysteines. Site-specifically conjugated ADCs have generallydemonstrated at least equivalent in vivo potency, improvedpharmacokinetics (PK), and an expanded therapeutic window compared towild-type conjugates. The first site-specific ADC in clinical trials,SGN-CD33A (Seattle Genetics), comprises a cleavable dipeptide linker(i.e., valine-alanine) and a DNA-cross-linking pyrrolobenzodiazepine(PBD) dimer as the drug, which is linked to a cysteine at heavy chainposition S239C in the Fc part of IgG1 mAb h2H12, having an averagedrug-to-antibody ratio (DAR) of 1.9 (Sutherland et al., Blood, 2013,122(8), 1455-1463). This low drug loading might not be suitable forevery linker drug or for every type of cancer. For less potent toxins orfor cancer types with a lower expression of target antigen, a higher DARmay be necessary. However, this may only become apparent in an advancedstage of development, when substantial time, effort and resources havebeen invested in the development of an antibody with one engineeredcysteine in the heavy or light chain. Furthermore, when it is apparentthat an average DAR of 1.9 is insufficient, additional cysteines must beengineered into the antibody, which requires new antibody development,and therefore new cell line development, which is a lengthy process.Additionally, determining the position of the one or more additionalcysteines is not a trivial matter, because e.g. not all positions enableconjugation or some positions might result in ADCs with unacceptablyhigh percentages of high molecular weight aggregates (HMW).

Therefore, new and more flexible conjugation processes, which can beused to easily tune the DAR of the resulting ADCs, without the necessityof undertaking a full new antibody development are still desired, aswell as ADCs having acceptable antigen binding properties,pharmacokinetics, in vivo efficacy, therapeutic index, and/or stability.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to a process for preparing antibody-drugconjugates and to antibody-drug conjugates wherein therapeutic moietiesare conjugated to one or more engineered cysteines as well as to one ormore reduced interchain cysteines via a cleavable or non-cleavablelinker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HIC profile of the vc-seco-DUBA ADC 1 d, conjugated via reducedinterchain cysteines only, DAR 2.5 (upper panel) and of the vc-seco-DUBAADC 1 c, conjugated via both engineered cysteines at position HC41 andreduced interchain cysteines, DAR 2.6 (lower panel).

FIG. 2. HIC profiles of vc-MMAE ADCs conjugated via both engineeredcysteines at position HC41 and reduced interchain cysteines withincreasing DAR, obtained using the process according to the inventionwith increasing amounts of TCEP.

FIG. 3. Caspase 3/7 assay of anti-PSMA-vc-seco-DUBA ADCs 3 a-3 dconjugated via both engineered cysteines at position HC41 and reducedinterchain cysteines with increasing DAR, obtained using the processaccording to the invention. Rituximab-vc-seco-DUBA is the non-bindingADC control.

FIG. 4. In vivo efficacy of the anti-5T4-vc-seco-DUBA ADCs 1 b (▾, DAR2.2) and 1 c (

, DAR 2.6) conjugated via both engineered cysteines at position HC41 andreduced interchain cysteines, ADC 1 d conjugated via reduced interchaincysteines only (random only,

, DAR 2.5) in the BT474 xenograft in ces1c^(e) KO nude mice.

FIG. 5. Full PK curves of the anti-5T4-vc-seco-DUBA ADC 1 c (DAR 2.6),conjugated via both engineered cysteines at position HC41 and reducedinterchain cysteines, and of the anti-5T4-vc-seco-DUBA ADC 1 d (DAR 2.5)that is conjugated via reduced interchain cysteines only.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to a process for preparing antibody-drugconjugates and to antibody-drug conjugates wherein therapeutic moietiesare conjugated to one or more engineered cysteines as well as to one ormore reduced interchain cysteines via a cleavable or non-cleavablelinker.

The term “antibody” as used throughout the present specification refersto a monoclonal antibody (mAb) comprising two heavy chains and two lightchains or an antigen binding fragment thereof, wherein at least oneinterchain disulfide bond is present, e.g. a Fab, Fab′ or F(ab′)₂fragment. Antibodies in accordance with the invention may be of anyisotype such as IgG, IgA or IgM antibodies. Preferably, the antibody isan IgG antibody, more preferably an IgG1 or IgG2 antibody. Theantibodies may be chimeric, humanized or human. Preferably, theantibodies are humanized. Even more preferably, the antibody is ahumanized or human IgG antibody, most preferably a humanized or humanIgG1 mAb. The antibody may have κ (kappa) or λ (lambda) light chains,preferably κ (kappa) light chains, i.e., a humanized or human IgG1-κantibody.

In humanized antibodies, the antigen-binding complementarity determiningregions (CDRs) in the variable regions of the HC and LC are derived fromantibodies from a non-human species, commonly mouse, rat or rabbit.These non-human CDRs may be placed within a human framework (FR1, FR2,FR3 and FR4) of the variable regions of the HC and LC. Selected aminoacids in the human FRs may be exchanged for the corresponding originalnon-human species amino acids to improve binding affinity, whileretaining low immunogenicity. Alternatively, selected amino acids of theoriginal non-human species FRs are exchanged for their correspondinghuman amino acids to reduce immunogenicity, while retaining theantibody's binding affinity. The thus humanized variable regions arecombined with human constant regions.

The terms “monoclonal antibody” and “mAb” as used herein refer to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally-occurring mutations that maybe present in minor amounts. Antibodies may be generated by immunizinganimals with a mixture of peptides representing the desired antigen.B-lymphocytes are isolated and fused with myeloma cells or single Blymphocytes were cultivated for several days in the presence ofconditioned medium and feeder cells. The myeloma or B lymphocytesupernatants containing the produced antibodies are tested to selectsuitable B lymphocytes or hybridomas. Monoclonal antibodies may beprepared from suitable hybridomas by the hybridoma methodology firstdescribed by Köhler et al., Nature, 1975, 256, 495-497. Alternatively,the RNA of suitable B-cells or lymphoma may be lysed, RNA may beisolated, reverse transcripted and sequenced. Antibodies may be maderecombinant DNA methods in bacterial, eukaryotic animal or plant cells(see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” mayalso be isolated from phage antibody libraries using the techniquesdescribed in the art, e.g. in Clackson et al., Nature 199, 1352, 624-628and Marks et al., J. Mol. Biol. 1991, 222, 581-597.

The term “cysteine engineered antibody” as used throughout the presentspecification refers to an antibody wherein one or more cysteineresidues have been introduced by genetic engineering techniques, eitherby replacing one or more amino acid residues of the antibody with acysteine, or by insertion of one or more cysteines into the primaryamino acid sequence.

The term “antibody-drug conjugate (ADC)” as used throughout the presentspecification refers to an antibody as defined hereinabove, to which oneor more therapeutic moieties are conjugated via a linker (linker drugs).The number of linker drugs conjugated to the antibody is usuallyreferred to as drug-to-antibody ratio (DAR). Conjugation processestypically yield a heterogeneous mixture of different ADC molecules, i.e.different DAR species having different drug-to-antibody ratios.Therefore, the term “ADC” also refers to such mixtures of DAR species.The term “average DAR” refers to the average DAR of the population ofsuch DAR species. As is well-known in the art, the DAR and drug loaddistribution can be determined, for example, by using hydrophobicinteraction chromatography (HIC) or reversed phase high-performanceliquid chromatography (RP-HPLC). HIC is particularly suitable fordetermining the average DAR.

The present invention relates to a process for preparing anantibody-drug conjugate comprising the steps of:

-   -   a. selectively reducing a cysteine-engineered antibody        comprising reacting an antibody comprising one or more        engineered cysteines at positions selected from heavy chain 40,        41, and 89 according to the Kabat numbering system, heavy chain        152, 153, 155, and 171 according to the Eu numbering system,        light chain 40 and 41 according to the Kabat numbering system,        and light chain 165 and 168 according to the Eu numbering        system, with a compound according to formula (I), (II), (III),        (IV), (V), (VI) or (VII)

-   -   or a salt thereof;    -   b. further reducing the selectively reduced antibody of step a.        with an interchain disulfide bond reducing agent; and    -   c. conjugating therapeutic moieties to the further reduced        antibody of step b. via cleavable or non-cleavable linkers.

In one embodiment of the present invention, the compound according toformula (I), (II), (III), (IV), (V), (VI) or (VII) is present in anamount of at least one molar equivalent per molar amount of engineeredcysteine. This means that in order to selectively reduce an antibodyhaving one engineered cysteine in the light chain or heavy chain, i.e.two engineered cysteines are present in the antibody, at least two molesof the compound are used per mole of antibody.

Preferred is a process wherein an amount of from 2 to 16 molarequivalents of the compound according to formula (I), (II), (III), (IV),(V), (VI) or (VII) per molar amount of engineered cysteine is used. Ifless than one molar equivalent per molar amount of engineered cysteineis used, complete reduction of all engineered cysteines is not achieved.The molar ratio of the compound per engineered cysteine affects the rateof reduction (uncapping) of the engineered cysteines, but it has noinfluence on the selectivity of the reduction.

Preferably, the invention relates to a process, wherein the antibodycomprises one or more engineered cysteines at positions selected fromheavy chain 40, 41, 152, and 153 and light chain 40, 41, and 165. Morepreferably, the antibody comprises one or more engineered cysteines atpositions selected from heavy chain 41 and light chain 40 and 41. Mostpreferably, the antibody to be used in accordance with the process ofthe invention comprises an engineered cysteine at heavy chain 41, i.e.HC41C.

In the context of the present invention, Kabat numbering is used forindicating the amino acid positions of engineered cysteines in the heavychain (HC) and light chain (LC) variable regions and Eu numbering isused for indicating the positions in the heavy chain and light chainconstant regions of the antibody. The expression “Kabat numbering”refers to the numbering system used for heavy chain variable domains orlight chain variable domains of the compilation of antibodies describedin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991).

The expression “Eu numbering” refers to the Eu index as in Kabat, E. A.et al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., NIHpublication no. 91-3242, pp. 662, 680, 689 (1991).

In accordance with the present invention, the cysteine-engineeredantibody may be prepared by using conventional molecular cloningtechniques or the heavy chain or light chain domain(s) of the antibodycarrying the cysteine mutation(s) can be synthesized as such using known(peptide or DNA) synthesis equipment and procedures. Typically,procedures similar to those disclosed in WO2015/177360 are used.

The compound according to formula (I), (II), (III), (IV), (V), (VI) or(VII) reduces selectively the engineered cysteines of the antibody inthe process of the invention. The compound according to formula (I) is2-(diphenylphosphino)benzenesulfonic acid and the compound according toformula (II) is 2-(dicyclohexylphosphino)-benzenesulfonic acid.Phosphines (I) and (II) are commercially available as e.g. the sulfonicacid or the sodium sulfonate salt from various suppliers, e.g.Sigma-Aldrich. The compound according to formula (III), (IV), (V), (VI)or (VII) may be prepared by procedures known in the art (analogous toprocedures disclosed in e.g., M. Bornand et al., Organometallics, 2007,26(14), 3585-3596 and T. Schultz et al., Synthesis, 2005, 6, 1005-1011).The compounds according to formulae (I), (II), (III), (IV), (V), (VI)and (VII) are readily deprotonated in aqueous solution and may formcorresponding sulfonate salts with cations present in the solution.Typical cations are, e.g., ammonium, tetramethylammonium,triethanolammonium, imidazolium, sodium, and potassium, i.e. cationspresent in commonly used buffer solutions for preparing ADCs.

Preferred in accordance with the present invention is a process whereinthe cysteine-engineered antibody is reacted with a compound according toformula (I), (II), (III), (V), (VI) or (VII), or a salt thereof. Morepreferred in accordance with the present invention is a process whereinthe cysteine-engineered antibody is reacted with a compound according toformula (I), (II) or (III), or a salt thereof. Even more preferred inaccordance with the present invention is a process wherein thecysteine-engineered antibody is reacted with a compound according toformula (I) or (II), or a salt thereof. Most preferred in accordancewith the present invention is a process wherein the cysteine-engineeredantibody is reacted with a compound according to formula (I) or a saltthereof.

The present inventors found inter alia that by using a compoundaccording to formula (I), (II), (III), (IV), (V), (VI) or (VII), thecapped engineered cysteines at specific positions in the Fab cavity thatis formed by the CH1, VH, VL and CL domains of the antibody areselectively reduced, while the interchain disulfide bonds formed by thenative cysteine residues of the antibody, i.e. the interchain cysteines,are left intact.

The interchain disulfide bond reducing agent to be used in accordancewith the process of the invention can be any agent suitable for reducinginterchain cysteine disulfide bonds. Suitable interchain disulfide bondreducing agents are well known to the skilled person, see e.g. R. E.Hansen et al., Analytical Biochemistry, 2009, 394, 147-158. Typically,they are water soluble and have a negative redox potential at pH 7. Thereducing agent may be a thiol or a phosphine. Suitable thiols include1,4-(dithiobutyl)-2-amine (DTBA), glutathione, cysteine,2-mercaptoethanol, 2-mercaptoethylamine, dithioerythritol (DTE), ordithiothreitol (DTT). Suitable phosphines includetris(3-sulfophenyl)phosphine, tris(2-carboxyethyl)phosphine (TCEP),tris(3-hydroxypropyl)phosphine (THPP), or tris(hydroxymethyl)phosphine.Preferred reducing agents are tris(3-sulfophenyl)phosphine, TCEP andDTT. The most preferred interchain disulfide bond reducing agent isTCEP.

The interchain disulfide bond reducing agent to be used in the processaccording to the invention is preferably present in an amount of morethan 0.1 molar equivalents per molar equivalent of antibody. Morepreferably, the interchain disulfide bond reducing agent is present inan amount of at least 0.25 molar equivalents per molar equivalent ofantibody. Even more preferably, the interchain disulfide bond reducingagent is present in an amount of from 0.25 to 3 or of from 0.25 to 2molar equivalents per molar equivalent of antibody. Even morepreferably, the interchain disulfide bond reducing agent is present inan amount of from 0.5 to 2 molar equivalents per molar equivalent ofantibody, most preferably 0.5 to 1 molar equivalent per molar equivalentof antibody.

The process of the present invention is performed under mild conditions,i.e. conditions under which the antibody is stable. Typically, theprocess in accordance with the present invention is performed in abuffered aqueous solution. The cysteine-engineered antibodies that areproduced in (mammalian) host cells and that are isolated and purifiedusing conventional equipment and procedures may need a buffer exchangein order to obtain the optimal conditions for the selective reductionprocess in accordance with the present invention. Suitable bufferedsolutions include phosphate-buffered saline (PBS), citrate, histidine,acetate and succinate buffered aqueous solutions. Additional salts andother solutes (e.g. sucrose, trehalose, EDTA) may be present in thebuffered aqueous solution. For the selective reduction step the bufferedsolutions preferably are acetate, histidine, or mixtures of acetate andhistidine buffered solutions.

The reaction temperature is typically in the range of from 0° C. to 40°C., the pH is typically in the range of from 4 to 8.

For the selective reduction step a., a pH in the range of from 4 to 7 ispreferred. More preferred for the selective reduction step is a pH inthe range of from 5 to 6.

Typically, unreacted/excess compound of formula (I), (II), (III), (IV),(V), (VI) or (VII) is removed before further reducing the selectivelyreduced antibody, typically by ultrafiltration/diafiltration (UF/DF),tangential flow filtration (TFF) or active carbon filtration.

The above-described reaction conditions for the selective reduction stepa. mainly affect the rate and degree of completion of the selectivereduction process and/or the stability of the antibody.

The interchain disulfide bond reduction step b. and subsequentconjugation step c. in accordance with the present invention may beperformed in a buffered aqueous solution, e.g. a citrate, a histidine ora succinate buffered aqueous solution, at a pH and temperature at whichthe antibody, the therapeutic moieties to be conjugated via cleavable ornon-cleavable linkers (linker drugs), and the resulting antibodyconjugate are stable. Typically, the pH is in the range of from 5 to 8,and the temperature is in the range of from 0° C. to 40° C.

For the interchain disulfide bond reduction and subsequent conjugationstep, a pH in the range of from 6 to 8 is preferred, more preferred is apH in the range of from 6.5 to 7.5, a pH in the range of from 6.8 to 7.3is most preferred. A preferred buffered aqueous solution in which theinterchain disulfide bond reduction step and subsequent conjugation stepin accordance with the present invention are performed, compriseshistidine.

Additional salts and other solutes (e.g. sucrose, trehalose, EDTA) maybe present in the buffered aqueous solution. In case the moiety to beconjugated to the antibody is poorly water soluble, e.g. in case of ahydrophobic linker drug, the moiety may be dissolved in an organic,water-miscible solvent. Suitable solvents include dimethyl sulfoxide(DMSO), dimethyl acetamide (DMA), propylene glycol, and ethylene glycol.

The resulting ADCs may be purified using standard methods known to theperson skilled in the art, e.g. active carbon filtration to removeexcess linker drug and hydrophobic interaction chromatography (HIC) toremove any unreacted antibody.

The ADCs prepared according to the process of the present invention maybe analyzed using analytical methods known in the art, e.g.high-performance liquid chromatography (HPLC), shielded hydrophobicphase HPLC (SHPC), hydrophobic interaction chromatography (HIC), andsize-exclusion chromatography (SEC).

Typically, the antibody to be used in accordance with the invention is amonospecific or bispecific antibody or antibody fragment comprising atleast one heavy chain and light chain variable region binding to atarget selected from the group consisting of annexin Al, B7H4, CA6, CA9,CA15-3, CA19-9, CA27-29, CA125, CA242, CCR2, CCR5, CD2, CD19, CD20,CD22, CD30, CD33, CD37, CD38, CD40, CD44, CD47, CD56, CD70, CD74, CD79,CD115, CD123, CD138, CD203c, CD303, CD333, CEA, CEACAM, CLCA-1, CLL-1,c-MET, Cripto, DLL3, EGFL, EGFR, EPCAM, EPh (e.g. EphA2 or EPhB3),endothelin B receptor (ETBR), FAP, FcRL5 (CD307), FGFR (e.g. FGFR3),FOLR1, GCC, GPNMB, HER2, HMW-MAA, integrin α (e.g. αvβ3 and αvβ5),IGF1R, TM4SF1 (or L6 antigen), Lewis A like carbohydrate, Lewis X, LewisY, LIV1, mesothelin, MUC1, MUC16, NaPi2b, Nectin-4, PD-1, PD-L1, PSMA,PTK7, SLC44A4, STEAP-1, 5T4 antigen (or TPBG, trophoblast glycoprotein),TF (tissue factor), TF-Ag, Tag72, TNFR, TROP2, VEGFR and VLA.

The linker to be used in accordance with the present invention shouldcomprise a functional group which can react with the thiol group of anuncapped engineered or reduced interchain cysteine, e.g. a maleimide ora haloacetyl group. The linker may be cleavable, e.g. comprising acleavable dipeptide), or non-cleavable. Suitable cleavable andnon-cleavable linkers are known in the art. For instance cleavablelinkers may comprise e.g. a valine-citrulline (vc) or a valine-alanine(va) moiety andsuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is anexample of a non-cleavable linker. Preferably, the linker used is acleavable linker.

The therapeutic moiety may be a drug, a toxin or a chelator for aradioactive isotope.

Preferred in accordance with the present invention is a process for thepreparation of an ADC, wherein the therapeutic moiety is a drug ortoxin.

A suitable therapeutic moiety includes a tubulin inhibitor (e.g. amaytansinoid, auristatin or tubulysin derivative), aribosome-inactivating protein (e.g. a saporin derivative), a DNA minorgroove binding agent (e.g. a duocarmycin or pyrrolobenzodiazepine (PBD)dimer or derivative), a DNA damaging agent (e.g. a PBD derivative), aDNA alkylating agent (e.g. a duocarmycin derivative), a DNAintercalating agent (e.g. a calicheamicin derivative), a DNAcrosslinking agent (e.g. a1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer derivative),an RNA polymerase inhibitor (e.g. an amanitin derivative), a DNAcleaving agent (e.g. a calicheamicin derivative) or an agent thatdisrupts protein synthesis or the function of essential cellularproteins (e.g. a topoisomerase I or II inhibitor (e.g. a camptothecinderivative), a proteasome inhibitor, a histone deacetylase inhibitor, anuclear export inhibitor, a kinase inhibitor, or an inhibitor of heatshock protein 90).

Preferably, the therapeutic moiety is a duocarmycin, a CBI dimer, acalicheamicin, a PBD, a PBD dimer, a maytansinoid, a tubulysin, acamptothecin, an amanitin, or an auristatin derivative. Most preferably,the therapeutic moiety is a duocarmycin derivative.

Examples of suitable therapeutic moieties include the duocarmycinseco-DUBA, the calicheamicin N-acetyl gamma calicheamicin dimethylhydrazide (CalichDMH), the PBD dimer SGD-1882, the maytansinoids DM1 andDM4, and the auristatins monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF).

Examples of combinations of linker and therapeutic moieties (linkerdrugs) to be used in the process according to the invention includevc-seco-DUBA (SYD980), mertansine, emtansine, ravtansine, mc-vc-PAB-MMAE(also abbreviated as mc-vc-MMAE or vc-MMAE), mc-MMAF, and mc-vc-MMAF.These abbreviations are well-known to the skilled artisan (see alsoWO2015/177360). The linker drug vc-seco-DUBA is disclosed inWO2011/133039 as compound 18b on p. 210, 11, 21-27. In a particularembodiment, the process according to the invention, comprises the step(step c.) of conjugating vc-seco-DUBA to the further reduced antibody ofstep b. as defined hereinabove, in which process preferably theinterchain disulfide bond reducing agent in step b. is present in anamount of from 0.5 to 1 molar equivalent per molar equivalent ofantibody.

Advantageously, the process according to the invention results in areaction product, i.e. an ADC, with a negligible amount of unreactedantibody (DAR0). Also the amount of high molecular weight (HMW) speciesis low in comparison with interchain cysteine-only conjugated ADCs,whereas the average DAR can be easily tuned by varying the amount ofinterchain disulfide bond reducing agent. The negligible amount of DAR0facilitates the purification process. Additionally, there is no loss ofantibody, which is the most costly part of an ADC. The process accordingto the invention is thus more efficient than the conjugation processesof the prior art.

The invention also relates to an ADC obtainable by the process describedhereinabove.

In one embodiment, the invention relates to an antibody-drug conjugate,wherein the antibody comprises one or more engineered cysteines atpositions selected from heavy chain 40, 41, and 89 according to theKabat numbering system, heavy chain 152, 153, 155, and 171 according tothe Eu numbering system, light chain 40 and 41 according to the Kabatnumbering system, and light chain 165 and 168 according to the Eunumbering system; wherein therapeutic moieties are conjugated via acleavable or non-cleavable linker to the one or more engineeredcysteines as well as to one or more reduced interchain cysteines of theantibody; and wherein the antibody-drug conjugate has an average DAR ofat least 2.0.

Preferably, the invention relates to an ADC as described hereinabove,wherein the antibody comprises one or more engineered cysteines atpositions selected from heavy chain 40, 41, 89, 152, and 153; and lightchain 40, 41, and 165. More preferably, the invention relates to an ADCas described hereinabove, wherein the antibody comprises one or moreengineered cysteines at positions selected from heavy chain 40 and 41,and light chain 40 and 41. Even more preferably, the invention relatesto an ADC as described hereinabove, wherein the antibody comprises oneor more engineered cysteines at positions selected from heavy chain 41,and light chain 40 and 41. Most preferably, the invention relates to anADC as described hereinabove, wherein the antibody comprises anengineered cysteine at position HC41.

Preferably, the invention relates to an ADC as described hereinabovewherein the therapeutic moieties conjugated via a cleavable ornon-cleavable linker to the one or more engineered cysteines as well asto one or more reduced interchain cysteines of the antibody are selectedfrom the group consisting of tubulin inhibitors (e.g. maytansinoid,auristatin or tubulysin derivatives), ribosome-inactivating proteins(e.g. saporin derivatives), DNA minor groove binding agents (e.g.duocarmycin or pyrrolobenzodiazepine (PBD) dimers or derivatives), DNAdamaging agents (e.g. PBD derivatives), DNA alkylating agents (e.g.duocarmycin derivatives), DNA intercalating agents (e.g. calicheamicinderivatives), DNA crosslinking agents (e.g.1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer derivatives),RNA polymerase inhibitors (e.g. amanitin derivatives), DNA cleavingagents (e.g. calicheamicin derivatives) or agents that disrupt proteinsynthesis or the function of essential cellular proteins (e.g.topoisomerase I or II inhibitors (e.g. camptothecin derivatives),proteasome inhibitors, histone deacetylase inhibitors, nuclear exportinhibitors, kinase inhibitors, or inhibitors of heat shock protein 90).

More preferred is an ADC according to the invention wherein thetherapeutic moieties are selected from the group consisting ofduocarmycin, CBI dimer, calicheamicin, PBD, PBD dimer, maytansinoid,tubulysin, camptothecin, amanitin, and auristatin derivatives. Mostpreferably, the therapeutic moieties are selected from the groupconsisting of duocarmycin derivatives or auristatin derivatives, such asMMAE and MMAF.

Even more preferably, the invention relates to an ADC, wherein theantibody comprises an engineered cysteine at position HC41, and whereinvc-seco-DUBA moieties or vc-MMAE moieties are conjugated to theengineered cysteines and to one or more reduced interchain cysteines andwherein the ADC has an average DAR of at least 2.0.

The optimal average DAR of an ADC is dependent on i.a. the type oftumour associated antigen, the type of antibody, the type of therapeuticmoiety, the type of linker and is dependent on the type of cancer. Theproperties of the antigen, e.g. the level of expression of the tumourassociated antigen on the surface of cancer cells, the distribution ofthe tumour associated antigen in healthy tissue as compared to diseasedtissue, and its internalization rate, all influence the optimal DAR ofthe ADC. Typically, ADCs according to the invention comprisinghydrophobic therapeutic moieties will have a lower optimal DAR than ADCsaccording to the invention comprising less hydrophobic therapeuticmoieties, because they form HMW species more easily than ADCs comprisingless hydrophobic therapeutic moieties. ADCs comprising less potenttherapeutic moieties might require a higher DAR in order to have thedesired efficacy.

The skilled person is able to determine the optimal DAR of any ADC foruse in the treatment of any type of cancer using conventional analyticaltechniques, such as SEC and HIC, combined with preclinical in vitrotests (e.g. cytotoxicity against cancer cell lines, using cytolysis ormembrane leakage assays) and in vivo experiments (e.g. efficacy inxenograft animal models).

In one preferred embodiment, the ADC according to the invention has anaverage DAR of at least 2.2. In a second preferred embodiment, the ADCaccording to the invention has an average DAR of from 2.0 to 6.0, morepreferably of from 2.2 to 6.0.

In one embodiment, the invention relates to an ADC, wherein the antibodycomprises an engineered cysteine at position heavy chain 41, and whereinvc-seco-DUBA moieties are conjugated to the engineered cysteines and toone or more reduced interchain cysteines and wherein the ADC has anaverage DAR of from 2.2 to 2.6.

In a second embodiment, the invention relates to an ADC, wherein theantibody comprises an engineered cysteine at position heavy chain 41 andwherein vc-MMAE moieties are conjugated to the engineered cysteines andto one or more reduced interchain cysteines and wherein the ADC has anaverage DAR of from 2.9 to 5.8.

Advantageously, the ADCs in accordance with the invention are lesshydrophobic than analogues with the same DAR which are conjugated viathe interchain cysteines only. This reduced hydrophobicity influencesthe pharmacokinetic properties of the ADC favorably, i.e. lessdeconjugation and increased efficacy were observed in vivo.

The present invention further relates to a pharmaceutical compositioncomprising an ADC as described hereinabove and one or morepharmaceutically acceptable excipients. Typical pharmaceuticalformulations of therapeutic proteins such as antibodies take the form oflyophilized cakes (lyophilized powders), which require (aqueous)dissolution (i.e. reconstitution) before intravenous infusion, or frozen(aqueous) solutions, which require thawing before use.

Typically, the pharmaceutical composition is provided in the form of alyophilized cake. Suitable pharmaceutically acceptable excipients forinclusion into the pharmaceutical composition (before freeze-drying) inaccordance with the present invention include buffer solutions (e.g.citrate, histidine or succinate containing salts in water),lyoprotectants (e.g. sucrose, trehalose), tonicity modifiers (e.g.sodium chloride), surfactants (e.g. polysorbate), and bulking agents(e.g. mannitol, glycine). Excipients used for freeze-dried proteinformulations are selected for their ability to prevent proteindenaturation during the freeze-drying process as well as during storage.

The present invention further relates to an ADC or pharmaceuticalcomposition as described hereinabove for use as a medicament, inparticular for use in the treatment or prevention of cancer, moreparticularly for use in the treatment of solid tumours andhaematological malignancies.

Examples

Materials and Methods

An anti-5T4 HC41C antibody comprising a HCVR amino acid sequence of SEQID NO: 1 as well as a LCVR amino acid sequence of SEQ ID NO:2 wasobtained using analogous procedures as the procedures described inWO2015/177360 using HC and LC leader sequences according to SEQ ID NO:3and 4.

The anti-PSMA cysteine-engineered HC41C antibody was obtained using thematerials and procedures described in WO2015/177360. Reagents andbuffers were procured from commercial suppliers. Compounds according toformula (I) (i.e. 2-(diphenylphosphino)benzenesulfonic acid) and (II)(i.e. 2-(dicyclohexylphosphino)benzenesulfonic acid),3-(diphenylphosphino)benzenesulfonic acid,4-(diphenylphosphino)benzenesulfonic acid, andtriphenylphosphine-3,3′,3″-trisulfonic acid were purchased fromSigma-Aldrich. The compounds according to formula (III), (IV), (V), (VI)and (VII) were prepared by lithiating benzenesulfonic acid, followed byreacting the lithiated benzenesulfonic acid with the appropriatedialkyl, diaryl or alkyl/aryl (chloro-) phosphine using proceduresanalogous to literature procedures, e.g. M. Bornand et al.,Organometallics, 2007, 26(14), 3585-3596 and T. Schultz et al.,Synthesis, 2005, 6, 1005-1011. The linker drug vc-seco-DUBA (SYD980) wassynthesized according to procedures as described in WO2011/133039.

Dual reduction and conjugation protocol

To a solution of HC41C engineered antibody (10 mg/ml, 100 mM histidine,pH 5) was added 2-(diphenylphosphino)benzenesulfonic acid (DPPBS,formula (I)) (32 molar equivalents per molar equivalent of theengineered antibody, 10 mM in water (MilliQ®)) and the resultingsolution was allowed to stand overnight at room temperature. Theselectively reduced antibody solution was rebuffered to 4.2 mMhistidine, 50 mM trehalose pH 6 (˜10 mg/ml) and treated with EDTA (4%v/v, 25 mM in water (MilliQ®)), TRIS (1% v/v, 1 M in water (MilliQ®), pH8) and TCEP (0.5-2 molar equivalents (depending on the antibody and thedesired DAR) per molar equivalent of the antibody, 3 mM in water(MilliQ®)). The resulting solution was incubated at room temperature for3.5 hours after which N,N-dimethyl acetamide (DMA) (final DMAconcentration 10% v/v) and linker drug (SYD980 or vcMMAE, ˜8 molarequivalents per molar equivalent of the antibody, 10 mM in DMA) wereadded. The resulting mixture was mixed in a roller mixer for 1 hour inthe absence of light, subsequently the mixture was allowed to stand atroom temperature overnight. Excess linker drug was removed via activatedcarbon filtration and the resulting clear solution was rebuffered to 4.2mM histidine, 50 mM trehalose pH 6 using centrifugal filtration. Theresulting ADCs were analyzed using HIC/SEC.

Protocol for Conjugation of Linker Drug to Interchain Cysteine OnlyUsing Heavy Chain 41C Engineered Antibody (Using N-EthylmaleimideCapping of the Engineered Cysteines (Heavy Chain 41C NEM CappedControl))

Wild-type conjugated control HC41C antibodies were synthesized by addingDPPBS (32 molar equivalents per molar equivalent of the antibody, 10 mMin water (MilliQ®)) to a solution of HC41C engineered antibody (10mg/ml, 100 mM histidine, pH 5). The resulting solution was allowed tostand overnight at room temperature The selectively reduced antibodysolution was rebuffered to 4.2 mM histidine, 50 mM trehalose pH 6 (˜10mg/ml) and treated with EDTA (4% v/v, 25 mM in water (MilliQ®)), TRIS(1% v/v, 1 M in water (MilliQ®), pH 8) after which DMA (final DMAconcentration was ˜10%) and N-ethylmaleimide (NEM, 10 mM in DMA, ˜3.5molar equivalents per molar equivalent of the antibody) were added tocap the cysteine at HC41. The resulting solution was incubated at roomtemperature for 2 hours after the mixture was rebuffered to 4.2 mMhistidine, 50 mM trehalose pH 6.

The NEM capped HC41C engineered antibody was treated with EDTA (4% v/v,25 mM in water (MilliQ®)), TRIS (1% v/v, IM in water (MilliQ®), pH 8)after which tris(2-carboxyethyl)phosphine (TCEP, 2 mM in water(MilliQ®), 1.33 equivalents) was added. The mixture was incubated atroom temperature for 2 hours after which DMA (final DMA concentrationwas ˜10%) and linker drug (SYD980 or vcMMAE) were added (10 mM in DMA,˜7 molar equivalents). After 3 hours activated carbon was added toremove excess linker drug and the solution was filtered and rebufferedto 4.2 mM histidine, 50 mM trehalose pH 6. The resulting ADCs wereanalyzed using HIC/SEC.

HIC

For analytical HIC, 5-10 μL of sample (1 mg/ml) was injected onto aTSKgel Butyl-NPR column (4.6 mm ID×3.5 cm L, Tosoh Bioscience, Cat. no.14947). The elution method consisted of a linear gradient from 100%Buffer A (25 mM sodium phosphate, 1.5 M ammonium sulphate, pH 6.95) to100% of Buffer B (25 mM sodium phosphate, pH 6.95, 20% isopropanol) at0.4 ml/min over 20 minutes. The column temperature was maintained at 25°C. A Waters Acquity H-Class UPLC system equipped with PDA-detector andEmpower software was used. Absorbance was measured at 214 nm to quantifythe average DAR and % DAR0.

SEC

Method A

5 μL of sample (1 mg/ml) was injected onto a TSKgel G3000SWXL column (5μm, 7.8 mm ID×30 cm L, Tosoh Bioscience, Cat. no. 08541) equipped with aTSKgel SWXL Guard column (7 μm, 6.0 mm ID×4.0 cm L, Tosoh Bioscience,Cat. no. 08543). The elution method consisted of elution with 100% 50 mMsodium phosphate, 300 mM NaCl, pH 7.5 at 0.6 ml/min for 30 minutes. Thecolumn temperature was maintained at 25° C. A Waters Acquity H-ClassUPLC system equipped with PDA-detector and Empower software was used.Absorbance was measured at 214 nm to quantify the amount of HMW species.

Method B

2.5 μL of sample (1 mg/ml) was injected onto an Acquity UPLC Protein BEHSEC column (200 Å, 1.7 μm, 4.6 mm ID×15 cm L, Waters, Cat. no.186005225). The elution method consisted of elution with a 1:1 mixtureof 200 mM sodium phosphate, pH 7.5 and 20% isopropanol in MilliQ® at 0.3ml/min for 10 minutes. The column temperature was maintained at 40° C. AWaters Acquity H-Class UPLC system equipped with PDA-detector andEmpower software was used. Absorbance was measured at 214 nm to quantifythe amount of HMW species.

Results

To various antibodies linker drugs were conjugated using the dualconjugation process. The analytical results are summarized in Table 1.The ADCs conjugated through both the engineered cysteines at HC41 andinterchain cysteines contain negligible amounts of DAR0 species and showless HMW species than the ADCs conjugated through interchain cysteinesonly. Also the retention times of all DAR peaks have decreased,indicative of less hydrophobic DAR species. FIG. 1 shows the differencein HIC profile of an anti-5T4-vc-seco-DUBA ADC conjugated throughinterchain cysteines only (upper panel) versus an anti-5T4-vc-seco-DUBAADC conjugated through both the engineered cysteines at HC41 and reducedinterchain cysteines (lower panel). The lower panel shows a morehomogenous ADC mixture. FIG. 2 shows the HIC profiles ofanti-5T4-vc-MMAE ADCs conjugated through both the engineered cysteinesat position HC41 and reduced interchain cysteines. The HIC profiles showthat the DAR of the ADC can easily be tuned by varying the amount ofTCEP in the interchain disulfide bond reduction step.

TABLE 1 Average ADC eq. TCEP DAR % HMW % DAR0 RT DAR4 anti-5T4(HC41C)-vc-seco-DUBA 1a 0 1.9 1.3^(a) 0.4 NA 1b 0.25 2.2 2.6^(a) 0.511.4 1c 0.5 2.6 4.9^(a) 0.3 11.4 1d* control 1.33 2.5 12.1^(a) 22.4 12.5anti-5T4 (HC41C)-vc-MMAE 2a 0.5 2.9 0.9^(b) 0.2 10.1 2b 1.0 3.8 0.8^(b)0.1 10.1 2c 2.0 5.8 0.6^(b) ^(c) 10.1 2d* control 1.16 1.7 0.6^(b) 32.312.0 anti-PSMA (HC41C)-vc-seco-DUBA 3a 0.25 2.0 2.7^(b) 1.9 11.3 3b 0.52.3 3.1^(b) 2.3 11.3 3c 0.75 2.8 3.2^(b) 1.5 11.3 3d 1.0 3.2 3.2^(b) 0.611.3 3e control^(d) 1.24 1.8 4.8^(b) 38.7 12.2 NA = not applicable*HC41C NEM capped control ^(a)SEC Method A ^(b)SEC Method B ^(c) amountunder detection limit ^(d)wt-anti-PSMA -vc-seco-DUBA

Apoptosis Assay

Experimental

LNCaP-C4.2 cells (15,000 cells/well) in complete growth medium (RPMI1640 media (Lonza; Walkersville, Md., USA) supplemented with 10% v/wFBS, qualified (Gibco-Life Technologies)) were plated in 96-well plates(90 μl/well). After 4 hours of incubation at 37° C., 5% CO₂, 10 μl ofeach anti-PSMA-vc-seco-DUBA ADC (3 a, 3 b, 3 c and 3 d) was added.Serial dilutions of each ADC were made in complete growth medium.Apoptosis was assessed after 72 hours using the luminogenic caspase-3/7substrate from the ApoTox-Glo™ Triplex assay kit of Promega Corporation(Madison, Wis., USA) according to the manufacturer's instructions.

Results

FIG. 3 shows that as the DAR of the anti-PSMA-vc-seco-DUBA ADCsincreases, the ADCs show increasingly higher caspase-3/7 activity.Higher caspase-3/7 activity is indicative of an increase in apoptosis.Apoptosis is therefore more stimulated by anti-PSMA-vc-seco-DUBA ADCshaving a higher DAR as indicated by the higher luminescence signal.

In Vivo Anti-Tumour Efficacy

Tumours were induced through subcutaneous injection of twenty million(2×10⁷) BT-474 cells in 200 μL RPMI 1640 medium containing matrigel(50:50, v:v, ref: 356237, BD Biosciences, France) into the right flankof female ces1c^(e) KO nude mice. BT-474 tumour cell implantation wasperformed 24 to 72 hours after a whole body irradiation with a γ-source(2 Gy, 60Co, BioMep, Dijon, France).

Mice were randomized over the treatment groups when the tumours reacheda mean volume of 200-300 mm³ until a group size of 7 animals wasachieved. A statistical test (analysis of variance) was performed toassure homogeneity between groups. Next the mice received a singleintravenous (tail vein) dose of either vehicle, 1 mg/kg anti-5T4(HC41C)-vc-seco-DUBA (1 b, DAR 2.2), 1 mg/kg anti-5T4(HC41C)-vc-seco-DUBA (1 c, DAR 2.6), 1 mg/kg anti-5T4(HC41C)-vc-seco-DUBA (1 d, interchain cysteine conjugation only, DAR2.5). Pilot studies have shown that in this model a 1 mg/kg dose ofanti-5T4 ADC will not lead to complete tumour remission, allowing theevaluation of the effect of modulations in ADC format. The day ofrandomization and start of treatment is indicated day 0 (D0).

The length and width of the tumours were measured twice a week withcalipers and the volume of the tumours was estimated by the formula:tumour volume=(width²×length)/2.

FIG. 4 shows the in vivo efficacy of the anti-5T4-vc-seco-DUBA ADCs 1 b(▾, DAR 2.2) and 1 c (

, DAR 2.6), both conjugated via both engineered cysteines at positionHC41 and reduced interchain cysteines, and ADC 1 d, conjugated viareduced interchain cysteines only (random only,

, DAR 2.5), in the 5T4-positive BT474 xenograft in ces1c^(e) KO nudemice. Immediately after dosing all ADCs slowed down tumour growth andafter ten days tumour volumes of the anti-5T4-vc-seco-DUBA ADCs 1 b and1c decreased (remission). In contrast, tumours in the mice treated withADC 1 d only slowed down in growth but tumour volumes did not reduce(tumour stasis). After 30 days, tumour regrowth was observed. Clearly,ADCs 1 b and 1c showed improved efficacy over ADC 1 d.

PK Studies

The treatment groups in this PK study consisted of 9 male and 9 femaleces1c^(e) KO nude mice. The mice received the ADCs 1 c or 1 d as asingle intravenous dose of 3 mg/kg. Blood samples were taken at multipletime point hours after dosing, cooled on ice water, and processed toplasma as soon as possible. Plasma samples were snap frozen in liquidnitrogen and stored at −80° C. until analysis. ADC (Conjugated Ab)levels and total antibody levels (Total Ab) levels in plasma werequantified using an ELISA-based method as described in Dokter et al.,Mol. Cancer Ther., 2014, 13, 2618-2629.

FIG. 5 shows the full PK curves of the ADC 1 c (DAR 2.6), conjugated viaboth engineered cysteines at position HC41 and reduced interchaincysteines, and of the ADC 1 d (DAR 2.5) that is conjugated via itsreduced interchain cysteines only. For ADC 1 c, the levels of ConjugatedAb and Total Ab decrease gradually after 96 hrs, which is indicative ofa stable ADC and little deconjugation. However, for ADC 1 d the level ofConjugated Ab decreases more steeply than the level of Total Ab. Thisphenomenon is indicative of deconjugation. ADC 1 c is thus lesssusceptible to deconjugation than ADC 1 d.

(HCVR anti-5T4 antibody HC41C) SEQ ID NO: 1 1EVQLVESGGD LAQPGGSLRL SCAVSGIDLS SYGMGWVRQA CGKGLEWVSI 51ISRNSVTYYA TWAKGRFTIS RDNSKNTVYL QMTSLRAEDT ALYFCARRAT 101YSGALGYFDI WGQGTLVTVS S (LCVR anti-5T4 antibody) SEQ ID NO: 2 1EIVMTQSPSS LSASVGDRVT ITCQASENIY STLAWYQQKP GKAPKLLIYD 51AFDLASGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GYSGTNVDNA 101 FGQGTKLEIK(HC rabbit leader sequence) SEQ ID NO: 3 1 MGWTLVFLFL LSVTAGVHS(LC rabbit leader sequence) SEQ ID NO: 4 1 MVSSAQFLGL LLLCFQGTRC

1. A process for preparing an antibody-drug conjugate comprising thesteps of: (a) selectively reducing a cysteine-engineered antibodycomprising reacting an antibody comprising one or more engineeredcysteines at positions selected from heavy chain 40, 41, and 89according to the Kabat numbering system, heavy chain 152, 153, 155, and171 according to the Eu numbering system, light chain 40 and 41according to the Kabat numbering system, and light chain 165 and 168according to the Eu numbering system, with a compound according toformula (I), (II), (III), (IV), (V), (VI) or (VII)

or a salt thereof; (b) further reducing the selectively reduced antibodyof step (a) with an interchain disulfide bond reducing agent; and (c)conjugating therapeutic moieties to the further reduced antibody of step(b) via cleavable or non-cleavable linkers.
 2. The process according toclaim 1, wherein the compound according to formula (I), (II), (III),(IV), (V), (VI) or (VII) is present in an amount of at least one molarequivalent per molar amount of engineered cysteine.
 3. The processaccording to claim 1, wherein the engineered cysteine antibody isreacted with a compound according to formula (I), (II) or (III), or asalt thereof.
 4. The process according to claim 1, wherein the antibodycomprises one or more engineered cysteines at positions selected fromheavy chain 40, 41, 152, and 153 and light chain 40, 41, and
 165. 5. Theprocess according to claim 4, wherein the antibody comprises one or moreengineered cysteines at positions selected from heavy chain 41 and lightchain 40 and
 41. 6. The process according to claim 1, wherein theinterchain disulfide bond reducing agent istris(3-sulfophenyl)phosphine, tris(2-carboxyethyl)phosphine ordithiothreitol.
 7. The process according to claim 1, wherein theinterchain disulfide bond reducing agent is present in an amount of morethan 0.1 molar equivalents per molar equivalent of antibody.
 8. Theprocess according to claim 1, wherein the antibody is a monospecific orbispecific antibody or antibody fragment comprising at least one heavychain and light chain variable region binding to a target selected fromthe group consisting of annexin Al, B7H4, CA6, CA9, CA15-3, CA19-9,CA27-29, CA125, CA242, CCR2, CCR5, CD2, CD19, CD20, CD22, CD30, CD33,CD37, CD38, CD40, CD44, CD47, CD56, CD70, CD74, CD79, CD115, CD123,CD138, CD203c, CD303, CD333, CEA, CEACAM, CLCA-1, CLL-1, c-MET, Cripto,DLL3, EGFL, EGFR, EPCAM, EPh (e.g. EphA2 or EPhB3), endothelin Breceptor (ETBR), FAP, FcRL5 (CD307), FGFR (e.g. FGFR3), FOLR1, GCC,GPNMB, HER2, HMW-MAA, integrin α (e.g. αvβ3 and αvβ5), IGF1R, TM4SF1 (orL6 antigen), Lewis A like carbohydrate, Lewis X, Lewis Y, LIV1,mesothelin, MUC1, MUC16, NaPi2b, Nectin-4, PD-1, PD-L1, PSMA, PTK7,SLC44 Å4, STEAP-1, 5T4 antigen (or TPBG, trophoblast glycoprotein), TF(tissue factor), TF-Ag, Tag72, TNFR, TROP2, VEGFR and VLA.
 9. Anantibody-drug conjugate obtainable by the process of claim
 1. 10. Anantibody-drug conjugate, wherein the antibody comprises one or moreengineered cysteines at positions selected from heavy chain 40, 41, and89 according to the Kabat numbering system, heavy chain 152, 153, 155,and 171 according to the Eu numbering system, light chain 40 and 41according to the Kabat numbering system, and light chain 165 and 168according to the Eu numbering system; wherein therapeutic moieties areconjugated via a cleavable or non-cleavable linker to the one or moreengineered cysteines as well as to one or more reduced interchaincysteines of the antibody; and wherein the antibody-drug conjugate hasan average DAR of at least 2.0.
 11. The antibody-drug conjugateaccording to claim 10, wherein the antibody comprises one or moreengineered cysteines at positions selected from heavy chain 41 and lightchain 40 and
 41. 12. The antibody-drug conjugate according to claim 10,having an average DAR of at least 2.2.
 13. The antibody-drug conjugateaccording to claim 10, having an average DAR of from 2.0 to 6.0.
 14. Apharmaceutical composition comprising an antibody-drug conjugateaccording to claim 10 and one or more pharmaceutically acceptableexcipients.
 15. (canceled)